Search

Your search keyword '"Cédric Laguri"' showing total 23 results
Start Over Author "Cédric Laguri" Language english
23 results on '"Cédric Laguri"'

1. Defining the function of OmpA in the Rcs stress response

Author

Kilian Dekoninck, Juliette Létoquart, Cédric Laguri, Pascal Demange, Robin Bevernaegie, Jean-Pierre Simorre, Olivia Dehu, Bogdan I Iorga, Benjamin Elias, Seung-Hyun Cho, and Jean-Francois Collet

Subjects
OmpA, RcsF, Rcs system, envelope stress, periplasm, lipopolysaccharide, Medicine, Science, Biology (General), QH301-705.5
Abstract

OmpA, a protein commonly found in the outer membrane of Gram-negative bacteria, has served as a paradigm for the study of β-barrel proteins for several decades. In Escherichia coli, OmpA was previously reported to form complexes with RcsF, a surface-exposed lipoprotein that triggers the Rcs stress response when damage occurs in the outer membrane and the peptidoglycan. How OmpA interacts with RcsF and whether this interaction allows RcsF to reach the surface has remained unclear. Here, we integrated in vivo and in vitro approaches to establish that RcsF interacts with the C-terminal, periplasmic domain of OmpA, not with the N-terminal β-barrel, thus implying that RcsF does not reach the bacterial surface via OmpA. Our results suggest a novel function for OmpA in the cell envelope: OmpA competes with the inner membrane protein IgaA, the downstream Rcs component, for RcsF binding across the periplasm, thereby regulating the Rcs response.

Published
2020
Full Text
View/download PDF

2. Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus

Author

Roberto Maya-Martinez, J. Andrew N. Alexander, Christian F. Otten, Isabel Ayala, Daniela Vollmer, Joe Gray, Catherine M. Bougault, Alister Burt, Cédric Laguri, Matthieu Fonvielle, Michel Arthur, Natalie C. J. Strynadka, Waldemar Vollmer, and Jean-Pierre Simorre

Subjects
peptidoglycan, NMR, X-ray crystallography, penicillin-binding protein 4, Staphylococcus aureus, cyclic muropeptides, Microbiology, QR1-502
Abstract

Peptidoglycan (PG) is an essential component of the cell envelope, maintaining bacterial cell shape and protecting it from bursting due to turgor pressure. The monoderm bacterium Staphylococcus aureus has a highly cross-linked PG, with ~90% of peptide stems participating in DD-cross-links and up to 15 peptide stems connected with each other. These cross-links are formed in transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) of classes A and B. Most S. aureus strains have three housekeeping PBPs with this function (PBP1, PBP2, and PBP3) but MRSA strains have acquired a third class B PBP, PBP2a, which is encoded by the mecA gene and required for the expression of high-level resistance to β-lactams. Another housekeeping PBP of S. aureus is PBP4, which belongs to the class C PBPs, and hence would be expected to have PG hydrolase (DD-carboxypeptidase or DD-endopeptidase) activity. However, previous works showed that, unexpectedly, PBP4 has transpeptidase activity that significantly contributes to both the high level of cross-linking in the PG of S. aureus and to the low level of β-lactam resistance in the absence of PBP2a. To gain insights into this unusual activity of PBP4, we studied by NMR spectroscopy its interaction in vitro with different substrates, including intact peptidoglycan, synthetic peptide stems, muropeptides, and long glycan chains with uncross-linked peptide stems. PBP4 showed no affinity for the complex, intact peptidoglycan or the smallest isolated peptide stems. Transpeptidase activity of PBP4 was verified with the disaccharide peptide subunits (muropeptides) in vitro, producing cyclic dimer and multimer products; these assays also showed a designed PBP4(S75C) nucleophile mutant to be inactive. Using this inactive but structurally highly similar variant, liquid-state NMR identified two interaction surfaces in close proximity to the central nucleophile position that can accommodate the potential donor and acceptor stems for the transpeptidation reaction. A PBP4:muropeptide model structure was built from these experimental restraints, which provides new mechanistic insights into mecA independent resistance to β-lactams in S. aureus.

Published
2019
Full Text
View/download PDF

3. Structure–function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ

Author

Sylvie Manuse, Nicolas L. Jean, Mégane Guinot, Jean-Pierre Lavergne, Cédric Laguri, Catherine M. Bougault, Michael S. VanNieuwenhze, Christophe Grangeasse, and Jean-Pierre Simorre

Subjects
Science
Abstract

Placement of the bacterial division site is crucial for the creation of identical daughter cells. Here, the authors solve the structure of the MapZ protein, which helps to position the cell division protein FtsZ at the cell centre, and further analyse the function of the protein in vivo.

Published
2016
Full Text
View/download PDF

4. The novel CXCL12gamma isoform encodes an unstructured cationic domain which regulates bioactivity and interaction with both glycosaminoglycans and CXCR4.

Author

Cédric Laguri, Rabia Sadir, Patricia Rueda, Françoise Baleux, Pierre Gans, Fernando Arenzana-Seisdedos, and Hugues Lortat-Jacob

Subjects
Medicine, Science
Abstract

BACKGROUND: CXCL12alpha, a chemokine that importantly promotes the oriented cell migration and tissue homing of many cell types, regulates key homeostatic functions and pathological processes through interactions with its cognate receptor (CXCR4) and heparan sulfate (HS). The alternative splicing of the cxcl12 gene generates a recently identified isoform, CXCL12gamma, which structure/function relationships remain unexplored. The high occurrence of basic residues that characterize this isoform suggests however that it could feature specific regulation by HS. METHODOLOGY/PRINCIPAL FINDINGS: Using surface plasmon resonance and NMR spectroscopy, as well as chemically and recombinantly produced chemokines, we show here that CXCL12gamma first 68 amino acids adopt a structure closely related to the well described alpha isoform, followed by an unfolded C-terminal extension of 30 amino acids. Remarkably, 60% of these residues are either lysine or arginine, and most of them are clustered in typical HS binding sites. This provides the chemokine with the highest affinity for HP ever observed (Kd = 0.9 nM), and ensures a strong retention of the chemokine at the cell surface. This was due to the unique combination of two cooperative binding sites, one strictly required, found in the structured domain of the protein, the other one being the C-terminus which essentially functions by enhancing the half life of the complexes. Importantly, this peculiar C-terminus also regulates the balance between HS and CXCR4 binding, and consequently the biological activity of the chemokine. CONCLUSIONS/SIGNIFICANCE: Together these data describe an unusual binding process that gives rise to an unprecedented high affinity between a chemokine and HS. This shows that the gamma isoform of CXCL12, which features unique structural and functional properties, is optimized to ensure its strong retention at the cell surface. Thus, depending on the chemokine isoform to which it binds, HS could differentially orchestrate the CXCL12 mediated directional cell kinesis.

Published
2007
Full Text
View/download PDF

5. LptB 2 FG is an ABC transporter with Adenylate Kinase activity regulated by LptC/A recruitment

Author

Jean-Pierre Simorre, Isabel Ayala, Paola Sperandeo, Moura Eccm, Alessandra Polissi, Tiago Baeta, Karine Giandoreggio-Barranco, Cédric Laguri, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), and Dept. of Pharmacological and Biomolecular Sciences University of Milano

Subjects
0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, ATPase, Adenylate kinase, ATP-binding cassette transporter, Transporter, Periplasmic space, Cell biology, 03 medical and health sciences, Transmembrane domain, chemistry.chemical_compound, 0302 clinical medicine, biology.protein, Inner membrane, Adenosine triphosphate, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

Lipopolysaccharide (LPS) is an essential glycolipid covering the surface of gram-negative bacteria. Its transport involves a dedicated 7 protein transporter system, the Lpt machinery, that physically spans the entire cell envelope. LptB2FG complex is an ABC transporter that hydrolyses Adenosine Triphosphate (ATP) to extract LPS from the inner membrane (IM). LptB2FG was extracted directly from IM with its original lipid environment by Styrene-Maleic acids polymers(SMA). SMA-LptB2FG in nanodiscs displays ATPase activity and a previously uncharacterized Adenylate Kinase (AK) activity. It catalyzes phosphotransfer between two ADP molecules to generate ATP and AMP. ATPase and AK activities of LptB2FG are both stimulated by the interaction on the periplasmic side with LptC and LptA partners and inhibited by the presence of LptC transmembrane helix. Isolated ATPase module (LptB) has weak AK activity in absence of LptF and LptG, and one mutation, that weakens affinity for ADP, has AK activity similar to that of fully assembled complex. LptB2FG is thus capable of producing ATP from ADP depending on the assembly of the Lpt bridge and the AK activity might be important to ensure efficient LPS transport in fully assembled Lpt system.

Published
2021

6. The lipopolysaccharide-transporter complex LptB2FG also displays adenylate kinase activity in vitro dependent on the binding partners LptC/LptA

Author

Cédric Laguri, Isabel Ayala, Karine Giandoreggio-Barranco, Jean-Pierre Simorre, Tiago Baeta, Paola Sperandeo, Elisabete C.C. M. Moura, Alessandra Polissi, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)

Subjects
Lipopolysaccharides, Models, Molecular, Accelerated Communication, nuclear magnetic resonance (NMR), ATPase, STD, saturation transfer difference, Adenylate kinase, ATP-binding cassette transporter, Biochemistry, adenylate kinase, Lipopolysaccharide transport, 03 medical and health sciences, 0302 clinical medicine, Adenosine Triphosphate, Escherichia coli, Inner membrane, AK, adenylate kinase, Molecular Biology, OM, outer membrane, 030304 developmental biology, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Chemistry, Escherichia coli Proteins, Membrane Proteins, Biological Transport, Cell Biology, Periplasmic space, LptAm, monomeric version of LptA, Transmembrane protein, NMR, Cell biology, IM, inner membrane, TBS, Tris-buffered saline, biology.protein, SMA, styrene-maleic acid, cell wall, LPS, lipopolysaccharide, ATP-Binding Cassette Transporters, ABC transporter, Bacterial outer membrane, Carrier Proteins, 030217 neurology & neurosurgery, lipopolysaccharide (LPS), Lpt, lipopolysaccharide transporter system, TM, transmembrane
Abstract

Lipopolysaccharide (LPS) is an essential glycolipid that covers the surface of gram-negative bacteria. The transport of LPS involves a dedicated seven-protein transporter system called the lipopolysaccharide transport system (Lpt) machinery that physically spans the entire cell envelope. The LptB2FG complex is an ABC transporter that hydrolyzes ATP to extract LPS from the inner membrane for transport to the outer membrane. Here, we extracted LptB2FG directly from the inner membrane with its original lipid environment using styrene-maleic acid polymers. We found that styrene-maleic acid polymers-LptB2FG in nanodiscs display not only ATPase activity but also a previously uncharacterized adenylate kinase (AK) activity, as it catalyzed phosphotransfer between two ADP molecules to generate ATP and AMP. The ATPase and AK activities of LptB2FG were both stimulated by the interaction on the periplasmic side with the periplasmic LPS transport proteins LptC and LptA and inhibited by the presence of the LptC transmembrane helix. We determined that the isolated ATPase module (LptB) had weak AK activity in the absence of transmembrane proteins LptF and LptG, and one mutation in LptB that weakens its affinity for ADP led to AK activity similar to that of fully assembled complex. Thus, we conclude that LptB2FG is capable of producing ATP from ADP, depending on the assembly of the Lpt bridge, and that this AK activity might be important to ensure efficient LPS transport in the fully assembled Lpt system.

Published
2021

7. Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability

Author

Jean-Pierre Simorre, Emanuela Erba, Cédric Laguri, Alessandra Romanelli, Alessandra M. Martorana, Elisabete C.C. M. Moura, Alessandra Polissi, Paola Sperandeo, Tiago Baeta, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano [Milano] (UNIMI), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Dipartimento di Scienze Farmaceutiche 'Pietro Pratesi' [Milan, Italie], and Università degli Studi di Milano = University of Milan (UNIMI)

Subjects
Microbiology (medical), thanatin, Antimicrobial peptides, lcsh:QR1-502, ATP-binding cassette transporter, Microbiology, lcsh:Microbiology, Bacterial cell structure, Lipopolysaccharide transport, 03 medical and health sciences, antimicrobial peptides, Inner membrane, 030304 developmental biology, Original Research, BACTH technique, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, Chemistry, lipopolysaccharide, Periplasmic space, Translocon, Lpt system, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, NMR, Cell biology, Bacterial outer membrane, bacterial cell wall
Abstract

International audience; The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal β-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC-LptA and LptA-LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC-LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria.

Published
2020

8. Staphylococcus aureus sacculus mediates activities of M23 hydrolases

Author

Alicja Razew, Cedric Laguri, Alicia Vallet, Catherine Bougault, Magdalena Kaus-Drobek, Izabela Sabala, and Jean-Pierre Simorre

Subjects
Science
Abstract

Abstract Peptidoglycan, a gigadalton polymer, functions as the scaffold for bacterial cell walls and provides cell integrity. Peptidoglycan is remodelled by a large and diverse group of peptidoglycan hydrolases, which control bacterial cell growth and division. Over the years, many studies have focused on these enzymes, but knowledge on their action within peptidoglycan mesh from a molecular basis is scarce. Here, we provide structural insights into the interaction between short peptidoglycan fragments and the entire sacculus with two evolutionarily related peptidases of the M23 family, lysostaphin and LytM. Through nuclear magnetic resonance, mass spectrometry, information-driven modelling, site-directed mutagenesis and biochemical approaches, we propose a model in which peptidoglycan cross-linking affects the activity, selectivity and specificity of these two structurally related enzymes differently.

Published
2023
Full Text
View/download PDF

9. Molecular recognition of Escherichia coli R1-type core lipooligosaccharide by DC-SIGN

Author

Ferran Nieto-Fabregat, Angela Marseglia, Michel Thépaut, Jean-Philippe Kleman, Massilia Abbas, Aline Le Roy, Christine Ebel, Meriem Maalej, Jean-Pierre Simorre, Cedric Laguri, Antonio Molinaro, Alba Silipo, Franck Fieschi, and Roberta Marchetti

Subjects
Microbiology, Structural biology, Science
Abstract

Summary: Due to their ability to recognize carbohydrate structures, lectins emerged as potential receptors for bacterial lipopolysaccharides (LPS). Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. We contributed to fill this gap by unveiling the molecular basis of the interaction between the lipooligosaccharide of Escherichia coli and the dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin (DC-SIGN). Specifically, a combination of different techniques, including fluorescence microscopy, surface plasmon resonance, NMR spectroscopy, and computational studies, demonstrated that DC-SIGN binds to the purified deacylated R1 lipooligosaccharide mainly through the recognition of its outer core pentasaccharide, which acts as a crosslinker between two different tetrameric units of DC-SIGN. Our results contribute to a better understanding of DC-SIGN-LPS interaction and may support the development of pharmacological and immunostimulatory strategies for bacterial infections, prevention, and therapy.

Published
2024
Full Text
View/download PDF

10. Acyl acceptor recognition by Enterococcus faecium l,d-transpeptidase Ldtfm

Author

Jean-Emmanuel Hugonnet, Sébastien Triboulet, Cédric Laguri, Jean-Pierre Simorre, Michel Arthur, Catherine M. Bougault, Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), U1138 equipe 12, Centre de Recherche Saint-Antoine (CR Saint-Antoine), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR58-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5), Laboratoire de Chimie et Biologie des Métaux ( LCBM - UMR 5249 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Centre de Recherche Saint-Antoine ( CR Saint-Antoine ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Institut de biologie structurale ( IBS - UMR 5075 ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ) -Université Joseph Fourier - Grenoble 1 ( UJF ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -IFR58-Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Paris Descartes - Paris 5 ( UPD5 ), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Centre de Recherche Saint-Antoine (UMRS893), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Ertapenem, Models, Molecular, Stereochemistry, Acylation, Enterococcus faecium, Peptidoglycan, beta-Lactams, Microbiology, Article, Substrate Specificity, chemistry.chemical_compound, Bacterial Proteins, Catalytic Domain, Binding site, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Binding Sites, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Active site, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Acceptor, Anti-Bacterial Agents, 3. Good health, Molecular Docking Simulation, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biochemistry, chemistry, Docking (molecular), Peptidyl Transferases, Mutagenesis, Site-Directed, biology.protein, Cysteine, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

Summary In Mycobacterium tuberculosis and ampicillin-resistant mutants of Enterococcus faecium, the classical target of β-lactam antibiotics is bypassed by l,d-transpeptidases that form unusual 3 → 3 peptidoglycan cross-links. β-lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l,d-transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium l,d-transpeptidase Ldtfm by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An nuclear magnetic resonance (NMR)-driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldtfm that were evaluated by site-directed mutagenesis and development of a cross-linking assay. Three residues are reported as critical for stabilisation of the acceptor in the Ldtfm active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with β-lactams.

Published
2015

11. C5-epimerase and 2-O-sulfotransferase associate in vitro to generate contiguous epimerized and 2-O-sulfated heparan sulfate domains

Author

Célia Halimi, Hugues Lortat-Jacob, Cédric Laguri, Aurélie Préchoux, Jean-Pierre Simorre, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-12-JSV5-0006,HepLibScreen,Elaboration chimioenzymatique de banques d'héparanes sulfates, Evaluation des spécificités d'interaction protéines/HS.(2012), Lortat-Jacob, Hugues, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, Jeunes Chercheuses et Jeunes Chercheurs - Elaboration chimioenzymatique de banques d'héparanes sulfates, Evaluation des spécificités d'interaction protéines/HS. - - HepLibScreen2012 - ANR-12-JSV5-0006 - JC - VALID, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Sulfotransferase, Magnetic Resonance Spectroscopy, Stereochemistry, Immunoprecipitation, Iduronic Acid, Iduronic acid, Plasma protein binding, Biochemistry, Substrate Specificity, chemistry.chemical_compound, Sulfation, Biosynthesis, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, chemistry.chemical_classification, Carbon Isotopes, General Medicine, Heparan sulfate, Surface Plasmon Resonance, Enzyme, chemistry, Molecular Medicine, Heparitin Sulfate, Sulfotransferases, Carbohydrate Epimerases
Abstract

International audience; Heparan sulfate (HS), a complex polysaccharide of the cell surface, is endowed with the remarkable ability to bind numerous proteins, and as such, regulates a large variety of biological processes. Protein binding depends on HS structure; however, in the absence of a template driving its biosynthesis, the mechanism by which protein binding sequences are assembled remains poorly known. Here, we developed chemically defined 13C-labeled substrate and NMR based experiments to simultaneously follow in real time the activity of HS biosynthetic enzymes and characterize the reaction products. Using this new approach, we report that the association of C5-epimerase and 2-O-sulfotransferase, which catalyze the production of iduronic acid and its 2-O-sulfation respectively, is necessary to processively generate extended sequences of contiguous IdoA2S-containing disaccharides, whereas modifications are randomly introduced when the enzymes are uncoupled. These data shed light on the mechanisms by which HS motifs are generated during biosynthesis. They support the view that HS structure assembly is controlled not only by the availability of the biosynthetic enzymes but also by their physical association, which in the case of the C5-epimerase and 2-O-sulfotransferase was characterized by an affinity of 80 nM as demonstrated by surface Plasmon Resonance experiments.

Published
2015

12. Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan

Author

Morgane Callon, Michel Arthur, Isabel Ayala, Sébastien Triboulet, Catherine M. Bougault, Cédric Laguri, Paul Schanda, Jean-Pierre Simorre, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Paris Descartes - Paris 5 (UPD5)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5), Institut de biologie structurale ( IBS - UMR 5075 ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ) -Université Joseph Fourier - Grenoble 1 ( UJF ), Université Paris Descartes - Paris 5 ( UPD5 ), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Centre de Recherche des Cordeliers ( CRC (UMR_S 872) ), and Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS )

Subjects
Models, Molecular, Peptidyl transferase, Pseudopeptidoglycan, Plasma protein binding, Bacillus subtilis, Peptidoglycan, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, Bacterial cell structure, Article, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Colloid and Surface Chemistry, Cell Wall, Catalytic Domain, Binding site, 030304 developmental biology, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, General Chemistry, biology.organism_classification, 0104 chemical sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Mutation, Peptidyl Transferases, biology.protein, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Protein Binding
Abstract

International audience; The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance to date no structure of a protein in complex with an intact bacterial peptidoglycan has been re-solved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spec-troscopy to derive for the first time an atomic model of an L,D-transpeptidase from Bacillussubtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains – the catalytic domain as well as the proposed peptidoglycan recognition domain – are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the impli-cation of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein-peptidogly -can complex paves the way for the design of new antibiotic drugs targeting L,D-transpeptidases. The strategy devel-oped here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis.

Published
2014

13. Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide

Author

Federica A. Falchi, Rebecca J. Taylor, Sebastian J. Rowe, Elisabete C. C. M. Moura, Tiago Baeta, Cedric Laguri, Jean-Pierre Simorre, Daniel E. Kahne, Alessandra Polissi, and Paola Sperandeo

Subjects
ABC transporter, ATPase, lipopolysaccharide transport, cell envelope, outer membrane biogenesis, proteoliposomes, Microbiology, QR1-502
Abstract

ABSTRACT Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of many Gram-negative bacteria, providing a barrier against the entry of toxic molecules. In Escherichia coli, LPS is exported to the cell surface by seven essential proteins (LptA-G) that form a transenvelope complex. At the inner membrane, the ATP-binding cassette (ABC) transporter LptB2FG associates with LptC to power LPS extraction from the membrane and transfer to the periplasmic LptA protein, which is in complex with the OM translocon LptDE. LptC interacts both with LptB2FG and LptADE to mediate the formation of the transenvelope bridge and regulates the ATPase activity of LptB2FG. A genetic screen has previously identified suppressor mutants at a residue (R212) of LptF that are viable in the absence of LptC. Here, we present in vivo evidence that the LptF R212G mutant assembles a six-protein transenvelope complex in which LptA mediates interactions with LptF and LptD in the absence of LptC. Furthermore, we present in vitro evidence that the mutant LptB2FG complexes restore the regulation of ATP hydrolysis as it occurs in the LptB2FGC complex to achieve wild-type efficient coupling of ATP hydrolysis and LPS movement. We also show the suppressor mutations restore the wild-type levels of LPS transport both in vivo and in vitro, but remarkably, without restoring the affinity of the inner membrane complex for LptA. Based on the sensitivity of lptF suppressor mutants to selected stress conditions relative to wild-type cells, we show that there are additional regulatory functions of LptF and LptC that had not been identified. IMPORTANCE The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli, by seven essential proteins. LptC is the unconventional regulatory subunit of the LptB2FGC ABC transporter, involved in coordinating energy production and LPS transport. Surprisingly, despite being essential for bacterial growth, LptC can be deleted, provided that a specific residue in the periplasmic domain of LptF is mutated and LptA is overexpressed. Here, we apply biochemical techniques to investigate the suppression mechanism. The data produced in this work disclose an unknown regulatory function of LptF in the transporter that not only expands the knowledge about the Lpt complex but can also be targeted by novel LPS biogenesis inhibitors.

Published
2023
Full Text
View/download PDF

14. Continuous evolution profiles for electronic-tongue-based analysis

Author

Yanxia Hou, Dayane Tada Batista, Roberto Calemczuk, Jamal Koubachi, Maria Genua, David Bonnaffé, Thierry Livache, Hugues Lortat-Jacob, Cédric Laguri, Pauline Fornarelli, Els Saesen, Arnaud Buhot, Structures et propriétés d'architectures moléculaire (SPRAM - UMR 5819), Institut Nanosciences et Cryogénie (INAC), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO), Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire pharmaceutique, Abivax, Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Groupe Structure et Activité des Glycosaminoglycanes (IBS-SAGAG ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
Analyte, Polymers, Stereochemistry, Aptamer, Biosensing Techniques, Plasma protein binding, 02 engineering and technology, Conjugated system, 010402 general chemistry, 01 natural sciences, Catalysis, Pattern Recognition, Automated, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, MESH: Heparitin Sulfate, [CHIM]Chemical Sciences, MESH: Pattern Recognition, Automated, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Surface plasmon resonance, Biochip, ComputingMilieux_MISCELLANEOUS, Chemistry, MESH: Electronics, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, General Medicine, Surface Plasmon Resonance, 021001 nanoscience & nanotechnology, Chemokine CXCL12, 0104 chemical sciences, MESH: Polymers, MESH: Surface Plasmon Resonance, Colloidal gold, Biophysics, Heparitin Sulfate, Electronics, MESH: Chemokine CXCL12, 0210 nano-technology, Biosensor, MESH: Biosensing Techniques
Abstract

Conventional biosensors and biochips rely on a “lock-andkey” recognition principle, for which specific ligands, such as aptamers, antibodies, or mimetics are needed. In contrast, electronic nose/tongue devices (eN/eT) use the differential binding of analytes, either alone or in mixtures, to an array of cross-reactive receptors (CRRs) whose combined responses create a characteristic pattern for each component. As a result, none of the CRRs need to be highly specific for any given analyte and the time-consuming design and generation of specific sensors may be circumvented. Based on this principle, CRR arrays associated with appropriate detection have been developed. For example, synthetic tetraphenylporphyrin derivatives, whose fluorescence quenching upon protein binding was used for detection, or a receptor library, integrating a peptide cavity coupled with indicator-uptake colorimetric detection, were developed for protein sensing above concentrations of ten micromolar. A sensor array composed of gold nanoparticles, grafted with different amino-functionalized thiols and conjugated with an anionic fluorescent polymer (poly(p-phenyleneethynylene, PPE), allowed detection of proteins in the low nanomolar range, as well as detection of bacteria or discrimination of normal cells from their cancerous counterparts. Furthermore, replacing PPE with green fluorescent protein led to a concentration-sensitive CRR array able to discriminate different proteins in human serum at physiologically relevant concentrations. However, though simplified with regard to conventional biosensors, the preparation of eN/eT sensing arrays still requires the design and synthesis 5–29 CRRs and conjugation to a ligand-independent detection system. The development of new eN/eTapplications would thus benefit from simplified production of the sensing elements. In this regard, eN applications take advantage of the differential diffusion of volatile biomarkers through films of a single sensing material in varying morphology and surface coverage. For an eT, we anticipated that the design and synthetic efforts could also be drastically reduced if the CRRs were prepared by selfassembly of different combinations of building blocks (BBs), that is easily accessible molecules displaying different physicochemical properties. In this way, simply mixing different BBs in varying and controlled proportions and allowing them to assemble, either alone or on a template, should lead to a collection of combinatorial cross-reactive receptors (CoCRRs) with evolutionary properties. Interestingly, a high diversity of CoCRRs can be produced from a restricted number of BBs: 11 with only two BBs mixed in concentrations varying from 0 to 100% in 10% increments and 66 by simply adding a third BB. Further generalization to n BBs and i% concentration increment leads to [(100/i)+ n 1]!/[(n 1)!(100/i)!] potential different CoCRRs. To assure 1) reproducible assembly; 2) close correlation between the CoCRRs topology and the precursor combination of BBs; 3) spatial encoding of the CoCRRs compositions, we reasoned that the BBs would best be combined through the formation of self-assembled monolayers (SAMs). Interestingly, if the gold surface of a surface plasmon resonance imaging (SPRi) prism was used for the formation of SAMs through thiol or disulfide bonds, a label-free, synchronous, parallel, and real-time observation of binding events on the CoCRRs array could also be performed (Figure 1). To investigate this hypothesis, we designed a model array inspired by the way cell-surface heparan sulfates (HS) recognize HS binding proteins (HSbps), an important group of extracellular mediators involved in many physiological and pathological processes. HS are negatively charged polysaccharides displaying a high degree of molecular diversity, which arises from the regulated generation of various epimerization and sulfation patterns during biosynthesis. According to cell type and activation state, HS chains with different negatively charged topologies are expressed, presumably to promote selective interactions with HSbps. [*] Dr. Y. Hou, M. Genua, Dr. D. Tada Batista, Dr. R. Calemczuk, Dr. A. Buhot, Dr. T. Livache SPrAM, UMR 5819 (CEA-CNRS-UJF-Grenoble 1), INAC/CEA-Grenoble 38054 Grenoble cedex 9 (France) E-mail: yanxia.hou-broutin@cea.fr thierry.livache@cea.fr

Published
2012

15. 13C-labeled heparan sulfate analogue as a tool to study protein/heparan sulfate interaction by NMR spectroscopy. Application to the CXCL12α chemokine

Author

Anne Imberty, Nicolas Sapay, Cédric Laguri, Bernhard Brutscher, Hugues Lortat-Jacob, Jean-Pierre Simorre, Pierre Gans, inconnu, Inconnu, Centre de Recherches sur les Macromolécules Végétales (CERMAV), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Carret, Michèle, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Chemokine, Magnetic Resonance Spectroscopy, Regulator, Molecular Probe Techniques, Oligosaccharides, Sequence (biology), 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, Glycosaminoglycan, 03 medical and health sciences, chemistry.chemical_compound, Colloid and Surface Chemistry, MESH: Heparitin Sulfate, MESH: Proteins, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, Carbon Isotopes, 0303 health sciences, Binding Sites, biology, MESH: Magnetic Resonance Spectroscopy, Proteins, MESH: Carbon Isotopes, General Chemistry, Heparan sulfate, Nuclear magnetic resonance spectroscopy, Oligosaccharide, Chemokine CXCL12, 0104 chemical sciences, MESH: Molecular Probe Techniques, chemistry, MESH: Binding Sites, biology.protein, Heparitin Sulfate, MESH: Chemokine CXCL12, Heparan sulfate analogue, MESH: Oligosaccharides
Abstract

International audience; Heparan sulfate (HS), a polysaccharide of the glycosaminoglycan family characterized by a unique level of complexity, has emerged as a key regulator of many fundamental biological processes. Although it has become clear that this class of molecules exert their functions by interacting with proteins, the exact modes of interaction still remain largely unknown. Here we report the engineering of a (13)C-labeled HS-like oligosaccharide with a defined oligosaccharidic sequence that was used to investigate the structural determinants involved in protein/HS recognition by multidimensional NMR spectroscopy. Using the chemokine CXCL12α as a model system, we obtained experimental NMR data on both the oligosaccharide and the chemokine that was used to obtain a structural model of a protein/HS complex. This new approach provides a foundation for further investigations of protein/HS interactions and should find wide application.

Published
2011

16. Dynamics of heparan sulfate explored by neutron scattering

Author

Cédric Laguri, Giuseppe Zaccai, Hugues Lortat-Jacob, Marion Jasnin, Judith Peters, Michael Marek Koza, Lambert van Eijck, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut Laue-Langevin (ILL), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and ILL

Subjects
Magnetic Resonance Spectroscopy, MESH: Molecular Structure, General Physics and Astronomy, Neutron scattering, 010402 general chemistry, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, Nuclear magnetic resonance, MESH: Heparitin Sulfate, Molecule, Scattering, Radiation, Neutron, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Physical and Theoretical Chemistry, MESH: Scattering, Radiation, 030304 developmental biology, Neutrons, 0303 health sciences, Molecular Structure, Chemistry, Scattering, MESH: Magnetic Resonance Spectroscopy, Nuclear magnetic resonance spectroscopy, Heparan sulfate, Nanosecond, 0104 chemical sciences, MESH: Neutrons, Chemical physics, Picosecond, Heparitin Sulfate
Abstract

International audience; The temperature dependence of atomic fluctuations in heparan sulfate was measured for different time-scales between the picosecond and the nanosecond. The data established the role of hydration for the emergence of high-amplitude motions at 200-240 K, and the higher resilience of the polysaccharide compared to proteins measured under similar conditions.

Published
2010

17. The CXCL12gamma chemokine displays unprecedented structural and functional properties that make it a paradigm of chemoattractant proteins

Author

Antonio Caruz, Ken Y. Chow, Karl Balabanian, Bernard Lagane, Françoise Baleux, Diego Franco, José L. Pablos, Cédric Laguri, Thierry Delaunay, Hugues Lortat-Jacob, Rabia Sadir, Fernando Arenzana-Seisdedos, Angélique Levoye, Isabelle Staropoli, Elena Lendinez, Patricia Rueda, Elena Izquierdo, Pathogénie Virale Moléculaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Departamento de Biología Experimental, Universidad de Jaén (UJA), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), UR 0420 - Station de pathologie végétale, Laboratoire de pathologie forestière, Institut National de la Recherche Agronomique (INRA)-Institut National de la Recherche Agronomique (INRA), Servicio de Reumatologia y Unidad de investigacion, Hospital de 12 Octubre, Chimie Organique (UCO), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), ANR, ANRS, ANR-05-BLAN-0271,CHEMOGLYCAN,STRUCTURAL AND FUNCTIONAL STUDIES OF SDF-1/CXCL12 INTERACTIONS WITH HEPARAN SULPHATE IN BOTH HOMEOSTASIS AND PATHOLOGY(2005), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Chimie Organique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Baleux, Françoise, and Programme non thématique - Appel à projets de recherche - STRUCTURAL AND FUNCTIONAL STUDIES OF SDF-1/CXCL12 INTERACTIONS WITH HEPARAN SULPHATE IN BOTH HOMEOSTASIS AND PATHOLOGY - - CHEMOGLYCAN2005 - ANR-05-BLAN-0271 - BLANC - VALID

Subjects
migration cellulaire, Chemokine, Extracellular matrix, Cell membrane, Mice, chemistry.chemical_compound, 0302 clinical medicine, Cell Movement, Cricetinae, Glycosaminoglycans, 0303 health sciences, Multidisciplinary, Microbiology and Parasitology, Heparan sulfate, Microbiologie et Parasitologie, Cell biology, Drug Combinations, medicine.anatomical_structure, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, 030220 oncology & carcinogenesis, Medicine, Proteoglycans, Physiology/Immune Response, Collagen, Research Article, Protein Binding, Receptors, CXCR4, Science, Immunology, Protein domain, CHO Cells, Biology, 03 medical and health sciences, Cricetulus, adhésion cellulaire, Extracellular, medicine, Animals, Humans, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, 030304 developmental biology, Chemotactic Factors, chemokine, Alternative splicing, Chemotaxis, Surface Plasmon Resonance, Chemokine CXCL12, Kinetics, chemistry, biology.protein, Laminin, pathologie humaine
Abstract

International audience; The CXCL12gamma chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12gamma is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12gamma through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12gamma both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12gamma strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12gamma one of the higher affinity for HS (K(d) = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12gamma to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12alpha. In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

Published
2008

18. Human Mismatch Repair Protein MSH6 Contains a PWWP Domain That Targets Double Stranded DNA

Author

Sophie Zinn-Justin, Bernard Gilquin, Marianne Axt, Joël Couprie, Isabelle Duband-Goulet, Nikolas Friedrich, Cédric Laguri, Pascal Belin, Isabelle Callebaut, CEA Laboratoire de Bilogie Structurale et Radiobiologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de minéralogie et de physique des milieux condensés (IMPMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Institut de Physique du Globe de Paris (IPG Paris)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie et Génétique Moléculaire, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie Structurale et Radiobiologie (LBSR), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
congenital, hereditary, and neonatal diseases and abnormalities, Magnetic Resonance Spectroscopy, HMG-box, DNA Repair, Somatic cell, Base Pair Mismatch, Molecular Sequence Data, Biology, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Conserved Sequence, 030304 developmental biology, 0303 health sciences, Binding Sites, Sequence Homology, Amino Acid, Mismatch Repair Protein, DNA, Molecular biology, Peptide Fragments, Proliferating cell nuclear antigen, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MSH6, DNA-Binding Proteins, Kinetics, chemistry, 030220 oncology & carcinogenesis, biology.protein, Chromatography, Gel, DNA mismatch repair, Colorectal Neoplasms, Sequence Alignment, Binding domain
Abstract

International audience; The eukaryotic mismatch repair (MMR) protein MSH6 exhibits a core region structurally and functionally similar to bacterial MutS. However, it possesses an additional N-terminal region (NTR), comprising a PCNA binding motif, a large region of unknown function and a nonspecific DNA binding fragment. Yeast NTR was recently described as an extended tether between PCNA and the core of MSH6 (1). In contrast, we show that human NTR presents a globular PWWP domain in the region of unknown function. We demonstrate that this PWWP domain binds double-stranded DNA, without any preference for mismatches or nicks, whereas its apparent affinity for single-stranded DNA is about 20 times lower. The S144I mutation, which in human MSH6 causes inherited somatic defects in MMR resulting in increased development of hereditary non polyposis colorectal cancer (2), is located in the DNA binding surface of the PWWP domain. However, it only moderately affects domain stability, and it does not perturb DNA binding in Vitro.

Published
2008

19. The Novel CXCL12γ Isoform Encodes an Unstructured Cationic Domain Which Regulates Bioactivity and Interaction with Both Glycosaminoglycans and CXCR4

Author

Patricia Rueda, Pierre Gans, Rabia Sadir, Fernando Arenzana-Seisdedos, Françoise Baleux, Hugues Lortat-Jacob, Cédric Laguri, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Pathogénie Virale Moléculaire, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Chimie Organique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), ANR, ANR-05-BLAN-0271,CHEMOGLYCAN,STRUCTURAL AND FUNCTIONAL STUDIES OF SDF-1/CXCL12 INTERACTIONS WITH HEPARAN SULPHATE IN BOTH HOMEOSTASIS AND PATHOLOGY(2005), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Baleux, Françoise, and Programme non thématique - Appel à projets de recherche - STRUCTURAL AND FUNCTIONAL STUDIES OF SDF-1/CXCL12 INTERACTIONS WITH HEPARAN SULPHATE IN BOTH HOMEOSTASIS AND PATHOLOGY - - CHEMOGLYCAN2005 - ANR-05-BLAN-0271 - BLANC - VALID

Subjects
Gene isoform, Cell type, Receptors, CXCR4, Magnetic Resonance Spectroscopy, Protein domain, Molecular Conformation, lcsh:Medicine, Plasma protein binding, Biology, Models, Biological, 03 medical and health sciences, chemistry.chemical_compound, Mice, 0302 clinical medicine, Cations, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, lcsh:Science, Biochemistry/Biomacromolecule-Ligand Interactions, 030304 developmental biology, Glycosaminoglycans, Regulation of gene expression, 0303 health sciences, Mice, Inbred BALB C, Multidisciplinary, Heparin, lcsh:R, Alternative splicing, Cell migration, Heparan sulfate, Surface Plasmon Resonance, Chemokine CXCL12, Protein Structure, Tertiary, chemistry, Biochemistry, Gene Expression Regulation, 030220 oncology & carcinogenesis, lcsh:Q, Research Article, Protein Binding
Abstract

International audience; BACKGROUND: CXCL12alpha, a chemokine that importantly promotes the oriented cell migration and tissue homing of many cell types, regulates key homeostatic functions and pathological processes through interactions with its cognate receptor (CXCR4) and heparan sulfate (HS). The alternative splicing of the cxcl12 gene generates a recently identified isoform, CXCL12gamma, which structure/function relationships remain unexplored. The high occurrence of basic residues that characterize this isoform suggests however that it could feature specific regulation by HS. METHODOLOGY/PRINCIPAL FINDINGS: Using surface plasmon resonance and NMR spectroscopy, as well as chemically and recombinantly produced chemokines, we show here that CXCL12gamma first 68 amino acids adopt a structure closely related to the well described alpha isoform, followed by an unfolded C-terminal extension of 30 amino acids. Remarkably, 60 % of these residues are either lysine or arginine, and most of them are clustered in typical HS binding sites. This provides the chemokine with the highest affinity for HP ever observed (Kd = 0.9 nM), and ensures a strong retention of the chemokine at the cell surface. This was due to the unique combination of two cooperative binding sites, one strictly required, found in the structured domain of the protein, the other one being the C-terminus which essentially functions by enhancing the half life of the complexes. Importantly, this peculiar C-terminus also regulates the balance between HS and CXCR4 binding, and consequently the biological activity of the chemokine. CONCLUSIONS/SIGNIFICANCE: Together these data describe an unusual binding process that gives rise to an unprecedented high affinity between a chemokine and HS. This shows that the gamma isoform of CXCL12, which features unique structural and functional properties, is optimized to ensure its strong retention at the cell surface. Thus, depending on the chemokine isoform to which it binds, HS could differentially orchestrate the CXCL12 mediated directional cell kinesis.

Published
2007

20. Solution structure and DNA binding of the effector domain from the global regulator PrrA (RegA) from Rhodobacter sphaeroides: insights into DNA binding specificity

Author

Cédric Laguri, Mary K. Phillips-Jones, Michael P. Williamson, Department of Molecular Biology and Biotechnology [Sheffield], University of Sheffield [Sheffield], and Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds

Subjects
Models, Molecular, HMG-box, Helix-turn-helix, Rhodobacter sphaeroides, Biology, Substrate Specificity, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Consensus Sequence, Genetics, Binding site, Phosphorylation, Nuclear Magnetic Resonance, Biomolecular, 030304 developmental biology, Helix-Turn-Helix Motifs, 0303 health sciences, Rhodobacter, Binding Sites, Base Sequence, 030306 microbiology, Articles, DNA, biology.organism_classification, Protein Structure, Tertiary, DNA binding site, DNA-Binding Proteins, Solutions, Response regulator, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], chemistry, Biochemistry, Trans-Activators, Nucleic Acid Conformation
Abstract

International audience; Prr/RegA response regulator is a global transcription regulator in purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus, and is essential in controlling the metabolic changes between aerobic and anaerobic environments. We report here the structure determination by NMR of the C-terminal effector domain of PrrA, PrrAC. It forms a three-helix bundle containing a helix-turn-helix DNA binding motif. The fold is similar to FIS protein, but the domain architecture is different from previously characterised response regulator effector domains, as it is shorter than any characterised so far. Alignment of Prr/RegA DNA targets permitted a refinement of the consensus sequence, which contains two GCGNC inverted repeats with variable half-site spacings. NMR titrations of PrrAC with specific and non-specific DNA show which surfaces are involved in DNA binding and suggest residues important for binding specificity. A model of the PrrAC/DNA complex was constructed in which two PrrAC molecules are bound to DNA in a symmetrical manner.

Published
2003

21. SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli

Author

Manuel Pazos, Katharina Peters, Adrien Boes, Yalda Safaei, Calem Kenward, Nathanael A. Caveney, Cedric Laguri, Eefjan Breukink, Natalie C. J. Strynadka, Jean-Pierre Simorre, Mohammed Terrak, and Waldemar Vollmer

Subjects
SPOR domain, cell division, peptidoglycan, peptidoglycan synthases, Microbiology, QR1-502
Abstract

ABSTRACT Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum. IMPORTANCE Escherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division.

Published
2020
Full Text
View/download PDF

22. Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability

Author

Elisabete C. C. M. Moura, Tiago Baeta, Alessandra Romanelli, Cedric Laguri, Alessandra M. Martorana, Emanuela Erba, Jean-Pierre Simorre, Paola Sperandeo, and Alessandra Polissi

Subjects
bacterial cell wall, lipopolysaccharide, Lpt system, thanatin, antimicrobial peptides, BACTH technique, Microbiology, QR1-502
Abstract

The outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal β-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC–LptA and LptA–LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC–LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria.

Published
2020
Full Text
View/download PDF

23. The CXCL12gamma chemokine displays unprecedented structural and functional properties that make it a paradigm of chemoattractant proteins.

Author

Patricia Rueda, Karl Balabanian, Bernard Lagane, Isabelle Staropoli, Ken Chow, Angelique Levoye, Cedric Laguri, Rabia Sadir, Thierry Delaunay, Elena Izquierdo, Jose Luis Pablos, Elena Lendinez, Antonio Caruz, Diego Franco, Françoise Baleux, Hugues Lortat-Jacob, and Fernando Arenzana-Seisdedos

Subjects
Medicine, Science
Abstract

The CXCL12gamma chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12gamma is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12gamma through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12gamma both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12gamma strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12gamma one of the higher affinity for HS (K(d) = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12gamma to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12alpha. In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

Published
2008
Full Text
View/download PDF

Catalog

Books, media, physical & digital resources
See catalog results